Cell-cell contacts play an important role in the normal function of endothelial cells. Contacts between cells not only act as a physical barrier that seals the vessel, but also alter the anatomical structure and biological function of these cells. Abnormalities in the nature of these contacts result in many pathologies of the vascular system, such as cancer metastasis and pulmonary edema. While it is known that cell-cell contacts change continually in response to the dynamic local environment in vivo, little is known about the cellular response to these changes in intercellular binding. Current in vitro models that promote or disrupt cell-cell contacts use pharmacologic or mechanical approaches that introduce confounding variables to the intracellular responses, and thus are not suited to address the causal relationship between cell-cell binding and cellular response. The proposed research intends to study the early (seconds to hours) stage response of endothelial cells to initiation of cell-cell contact. It hypothesizes that the initial contact between contacts plays a causal role in determining the sequence of events that ultimately lead to cell-cell junctional formation, intracellular signaling, cytoskeletal anchoring and reorganization, and cell differentiation. The behaviors to be examined are the rearrangement of cytoskeleton, the relocalization of membrane proteins, and the activation of signaling pathways upon the initial and subsequent binding between cells. This research will develop a general methodology based on the self-assembly of microscale, free-standing substrates on which cells are attached, to introduce physical cell-cell contact at specific times and without alteration of cell-matrix interactions. Identification of the events that occur within the cell upon initiation of intercellular binding will help to advance the understanding of the molecular and structural bases of normal and pathologic vessel formation, and may lead to the development of effective therapies to treat certain vascular diseases.